It has been generally recognized that the intracellular expression of naturally secreted eukaryotic proteins in microorganisms such as bacteria or yeast, as an expressed, mature polypeptide will frequently contain an additional, obligatory, initiation codon-derived methionine residue at the amino terminus. In many situations the extra amino acid is not detrimental, yet if these proteins are used for pharmaceutical indications, immunogenicity problems associated with this additional residue can be problematic.
Subsequent to the development of the initial intracellular expression systems, where the "methionine problem" was first encountered, several methods for circumventing this problem were developed. First and foremost, the viability of heterologous secretion systems for bacteria, yeast, filamentous fungi, and insect and mammalian cells was demonstrated. Indeed, for complex high molecular weight and glycosylated proteins, mammalian cell secretion systems have been essential. Heterologous secretion has, however, tended to be much lower yielding when compared with intracellular expression, and in some cases, secretion systems have failed altogether to generate relevant quantities of recombinant protein.
Secondly, in vitro systems involving chemical removal of methionine by cyanogen bromide and specific processing of fusion proteins with aminopeptidases, enterokinase, collagenase, and factor Xa have been developed in order to retain the high yields often associated with intracellular expression systems. It would be, however, desirable to avoid the use of the additional processing steps required for the cleavage reaction.
Ubiquitin (Ub), a highly conserved 76 residue protein, is found in eukaryotes either free or covalently joined via its carboxy-terminal glycine residue to a variety of cytoplasmic, nuclear, and integral membrane proteins. The coupling of ubiquitin to such proteins serves to target that protein as a proteolytic substrate for degradation. An important component of the degradation signal in a short-lived protein is the protein's amino-terminal residue (Bachmair et al., (1986) Science 234:179-186). The degradative pathway whose initial steps involve amino-terminal recognition of proteolytic substrates is called the N-end rule pathway, to distinguish it from other proteolytic pathways and also from other ubiquitin-dependent processes, some of which may not involve degradation of target proteins.
Varshavsky and coworkers (Varshavsky et al., (1988) in Ubiguitin (Rechsteiner, ed) pp 287-324, Plenum Press, New York; Varshavsky et al., (1989) in Yeast Genetic Engineering (Barr, Brake and Valenzuela, eds) pp 109-143, Butterworths, N.Y.; and PCT WO88/02406, published Apr. 7, 1988) have shown that ubiquitin may be utilized for the production of recombinant proteins with specifically engineered amino termini. Initially, the production of bacterial beta-galactosidase derivatives, and murine dihydrofolate reductases (DHFRS) that differed exclusively at their amino-terminal residues lead to the definition of the N-end rule. According to this general rule, specific amino acids can be ranked according to the degree of stabilization, or destabilization, that they confer upon proteins when positioned at their amino termini. Specifically, in Saccharomyces cerevisiae, any of the stabilizing amino-terminal residues (Met, Gly, Val, Pro, Cys, Ala, Ser, Thr) confers a long (greater than 20 hr) half-life on the test protein beta-galactosidase, whereas destabilizing amino-terminal residues (Ile and Glu, about 30 min; His, Tyr and Gln, about 10 min; Asp, Asn, Phe, Leu, Trp and Lys, about 3 min; and Arg, about 2 min) confer on beta-galactosidase half-lives from less than 3 min to 30 min.
Bachmair et al., supra in their N-end rule work described above, showed the capacity of the endogenous yeast processing enzyme to accurately cleave Ub from heterologous fusion proteins containing any of the 20 amino acids at the Ub-protein junction. Only in the case of proline was this process slow enough to observe Ub-fusion intermediates. For accurate determination of half-lives of the amino-terminally mutated test proteins (a beta-galactosidase derivative and dihydrofolate reductase) it was important to avoid such complications as inclusion body formation and this was achieved by the use of a relatively weak promoter system.
More recently, Butt et al., (1988) J Biol Chem 263:16364-16371 describe studies of ubiquitin fused with a homologous yeast protein, metallothionein. The hybrid gene is under control of the yeast metallothionein promoter, a promoter of intermediate strength. Ecker et al., (1989) J Biol Chem 264(13):7715-7719 also describe the use of the yeast metallothionein promoter to increase ubiquitin fused gene expression of G.sub.8 alpha, sCD4, and the protease domain of human urokinase in yeast while Butt et al., Proc Natl Acad Sci USA 86:2540-2544 (1989) describe a similar ubiquitin expression system developed for use in E. coli.
It would be desirable to develop a yeast expression vector system, preferably an inducible system, for expression of ubiquitin fusion proteins with simultaneous in vivo processing to yield authentic biologically active proteins having destabilizing amino terminal residues.
It would also be desirable to develop a general method using this vector system for quantitative processing of ubiquitin fusions to produce high expression levels of the desired heterologous protein in a yeast host.
In furtherance of these objectives, Barr et al., (1988) Yeast 4:S24 (Abstract) and Sabin et al., (1989) Biotechnology 7:705-709 have extended the observations by Varshavsky and, using strong and regulatable promoters, have produced in yeast high levels of heterologous eukaryotic proteins. Surprisingly, all of the proteins initiate with residues that are known to be destabilizing, yet, with one exception, each of the proteins expressed using the ubiquitin vector system were found to be correctly processed. The results of this work are reproduced herein.